Amatoxins are cyclic peptides composed of 8 amino acids that are found in Amanita phalloides mushrooms (see FIG. 1). Amatoxins specifically inhibit the DNA-dependent RNA polymerase II of mammalian cells, and thereby also the transcription and protein biosynthesis of the affected cells. Inhibition of transcription in a cell causes stop of growth and proliferation. Though not covalently bound, the complex between amanitin and RNA-polymerase II is very tight (KD=3 nM). Dissociation of amanitin from the enzyme is a very slow process, thus making recovery of an affected cell unlikely. When the inhibition of transcription lasts too long, the cell will undergo programmed cell death (apoptosis).
The use of amatoxins as cytotoxic moieties for tumour therapy had already been explored in 1981 by coupling an anti-Thy 1.2 antibody to α-amanitin using a linker attached to the indole ring of Trp (amino acid 4; see FIG. 1) via diazotation (Davis & Preston, Science 213 (1981) 1385-1388). Davis & Preston identified the site of attachment as position 7′. Morris & Venton demonstrated as well that substitution at position 7′ results in a derivative, which maintains cytotoxic activity (Morris & Venton, Int. J. Peptide Protein Res. 21 (1983) 419-430).
Patent application EP 1 859 811 A1 (published Nov. 28, 2007) described conjugates, in which the γ C-atom of amatoxin amino acid 1 of β-amanitin was directly coupled, i.e. without a linker structure, to albumin or to monoclonal antibody HEA125, OKT3, or PA-1. Furthermore, the inhibitory effect of these conjugates on the proliferation of breast cancer cells (MCF-7), Burkitt's lymphoma cells (Raji) and T-lymphoma cells (Jurkat) was shown. The use of linkers was suggested, including linkers comprising elements such as amide, ester, ether, thioether, disulfide, urea, thiourea, hydrocarbon moieties and the like, but no such constructs were actually shown, and no more details, such as attachment sites on the amatoxins, were provided.
Patent applications WO 2010/115629 and WO 2010/115630 (both published Oct. 14, 2010) describe conjugates, where antibodies, such as anti-EpCAM antibodies such as humanized antibody huHEA125, are coupled to amatoxins via (i) the γ C-atom of amatoxin amino acid 1, (ii) the 6′ C-atom of amatoxin amino acid 4, or (iii) via the δ C-atom of amatoxin amino acid 3, in each case either directly or via a linker between the antibody and the amatoxins. The suggested linkers comprise elements such as amide, ester, ether, thioether, disulfide, urea, thiourea, hydrocarbon moieties and the like. Furthermore, the inhibitory effects of these conjugates on the proliferation of breast cancer cells (cell line MCF-7), pancreatic carcinoma (cell line Capan-1), colon cancer (cell line Colo205), and cholangiocarcinoma (cell line OZ) were shown.
Amatoxins can be isolated from collected Amanita phalloides mushrooms fruit bodies, or from pure cultures (Zhang P, Chen Z, Hu J, Wei B, Zhang Z, and Hu W, Production and characterization of Amanitin toxins from a pure culture of Amanita exitialis, FEMS Microbiol Lett. 2005 Nov. 15; 252(2):223-8. Epub 2005 Sep. 15). However, the amounts of amatoxins that can be obtained are rather low (in the range of about 0.3-3 mg/g dry matter from natural fruit bodies, and about 10% thereof from pure culture) and the flexibility for further modifying the naturally occurring amatoxin variants is limited (see references discussed in [003]-[005] and references cited therein).
Alternatively, amatoxins can be obtained from fermentation using a basidiomycete (Muraoka S, and Shinozawa T., Effective production of amanitins by two-step cultivation of the basidiomycete, Galerina fasciculata GF-060, J Biosci Bioeng. 2000; 89(1):73-6; the reported yield was about 5 mg/l culture) or A. fissa (Guo X W, Wang G L, and Gong J H, Culture conditions and analysis of amanitins on Amanita spissa, Wei Sheng Wu Xue Bao. 2006 June; 46(3):373-8; the reported yield was about 30 μg/l culture). Again, yields are low, and flexibility for further modifying the naturally occurring amatoxin variants is limited as well.
Finally, amatoxins have been prepared by partial or total synthesis (e.g. Zanotti G, Möhringer C, and Wieland T., Synthesis of analogues of amaninamide, an amatoxin from the white Amanita virosa mushroom, Int J Pept Protein Res. 1987 October; 30(4):450-9; Zanotti G, Wieland T, Benedetti E, Di Blasio B, Pavone V, and Pedone C., Structure-toxicity relationships in the amatoxin series. Synthesis of 5-deoxy[gamma(R)-hydroxy-Ile3]-amaninamide, its crystal and molecular structure and inhibitory efficiency, Int J Pept Protein Res. 1989 September; 34(3):222-8; Zanotti G, Petersen G, and Wieland T., Structure-toxicity relationships in the amatoxin series. Structural variations of side chain 3 and inhibition of RNA polymerase II, Int J Pept Protein Res. 1992 December; 40(6):551-8; Zhao et al., Synthesis of a Cytotoxic Amanitin for Biorthogonal Conjugation, ChemBioChem 16 (2015) 1420-1425).
It is worth noting that Zanotti et al., 1989 (loc. cit.) reports the synthesis of the biologically inactive S-deoxo-γ(R)-hydroxy-isoleucin-amaninamide, while the synthesis of the active enantiomer has apparently not been successful.
Furthermore, Zhao et al. (loc. cit) report the synthesis of a γ-amaninamide derivative starting from 4(S)-4-hydroxyisoleucin. The authors, however, identified that the resulting γ-amaninamide derivative was only of negligible toxicity and thus taught away from further developing such γ-amaninamide-based structures.
While the use of fully-synthetic routes to amatoxins may offer an option for the supply of larger quantities of amatoxins required for therapeutic uses, and may offer the construction of a variety of novel α- or β-amatoxin variants by using appropriate starting materials as building blocks, the approaches pursued in the past had been limited by the fact that an essential building block, γ,δ-dihydroxyisoleucine, or a synthon therefor, had not been available as pure diastereomer. While WO 2014/009025 describes a novel synthon for γ,δ-dihydroxyisoleucine and its use for the synthesis of amatoxins, such approach is nevertheless expensive and labor-extensive.